Appl Microbiol Biotechnol (2012) 95:991–999
DOI 10.1007/s00253-012-3945-z
APPLIED MICROBIAL AND CELL PHYSIOLOGY
Lactobacillus reuteri CRL 1101 highly produces mannitol
from sugarcane molasses as carbon source
Maria Eugenia Ortiz & María José Fornaguera &
Raúl R. Raya & Fernanda Mozzi
Received: 31 December 2011 / Revised: 2 February 2012 / Accepted: 3 February 2012 / Published online: 22 February 2012
# Springer-Verlag 2012
Abstract Mannitol is a natural polyol extensively used in
the food industry as low-calorie sugar being applicable for
diabetic food products. We aimed to evaluate mannitol
production by Lactobacillus reuteri CRL 1101 using sugar-
cane molasses as low-cost energy source. Mannitol forma-
tion was studied in free-pH batch cultures using 3–10%
(w/v) molasses concentrations at 37 °C and 30 °C under static
and agitated conditions during 48 h. L. reuteri CRL 1101 grew
well in all assayed media and heterofermentatively converted
glucose into lactic and acetic acids and ethanol. Fructose was
used as an alternative electron acceptor and reduced it to
mannitol in all media assayed. Maximum mannitol concen-
trations of 177.7±26.6 and 184.5±22.5 mM were found
using 7.5% and 10% molasses, respectively, at 37 °C
after 24-h incubation. Increasing the molasses concentration
from 7.5% up to 10% (w/v) and the fermentation period up to
48 h did not significantly improve mannitol production.
In agitated cultures, high mannitol values (144.8 ±
39.7 mM) were attained at 8 h of fermentation as
compared to static ones (5.6 ± 2.9 mM), the highest
mannitol concentration value (211.3±15.5 mM) being found
after 24 h. Mannitol 2-dehydrogenase (MDH) activity was
measured during growth in all fermentations assayed; the
highest MDH values were obtained during the log growth
phase, and no correlation between MDH activities and man-
nitol production was observed in the fermentations performed.
L. reuteri CRL 1101 successfully produced mannitol from
Maria Eugenia Ortiz and María José Fornaguera contributed equally to
the article.
M. E. Ortiz : M. J. Fornaguera : R. R. Raya : F. Mozzi (*)
Centro de Referencia para Lactobacilos (CERELA)-CONICET,
Chacabuco 145,
San Miguel de Tucumán 4000, Argentina
e-mail: fmozzi@cerela.org.ar
sugarcane molasses being a promising candidate for microbial
mannitol synthesis using low-cost substrate.
Keywords Mannitol . Lactobacillus . Lactic acid bacteria .
Sugarcane molasses
Introduction
Mannitol is the most abundant occurring polyol in nature; it
is found in fruits and vegetables and is naturally produced
by yeasts, fungi, algae and bacteria. Mannitol has multiple
applications in the pharmaceutical, medical, chemical and
food industries (Monedero et al. 2010; Saha and Racine
2011; Vrancken et al. 2010). In food production, mannitol
is mainly employed as sweetener and non-metabolisable
low-calorie sugar (referred as E421 in the European Union)
being applicable for diabetic food products (Livesey 2003).
As food ingredient, it is also used in ‘breath-freshening’
products due to its negative enthalpy and capability to mask
undesirable tastes of many compounds (Debord et al. 1987).
In addition, it has been claimed to display other health-
promoting (anti-oxidant, anticariogenic) effects (Shen et al.
1997). Also, Liong and Shah (2005) found that mannitol
could act as prebiotic as it was successfully fermented
towards the production of organic acids by a Lactobacillus
acidophilus strain.
Mannitol can be produced by chemical, enzymatic or
microbiological processes. Nowadays, industrial production
is carried out by catalytic hydrogenation of a fructose/glu-
cose mixture where mannitol is produced together with
sorbitol, isomer which has less interesting properties and
lower market price (Carvalheiro et al. 2011). Separation of
sorbitol is rather difficult; thus, different alternatives to the
current production method have been sought (Vandamme
992
and Soetaert 1995). In the last years, efforts have been made
to produce mannitol through fermentations; several lactic
acid bacteria (LAB) have been used with this purpose
(Carvalheiro et al. 2011; Racine and Saha 2007; Saha
2006a, b; von Weymarn 2002; von Weymarn et al. 2003) as
these microorganisms are suited for polyol production since
they display fermentative metabolism associated with an
important redox modulation and limited biosynthetic capacity
(Monedero et al. 2010).
LAB are involved in numerous spontaneous fermented
foods and have been extensively used in the food industry as
they contribute to the overall quality of the fermented prod-
ucts improving their organoleptic, technological and nutri-
tional properties as well as prolonging their shelf-life
(Hugenholtz and Smid 2002). Mannitol-producing LAB
can be used in situ to produce naturally enriched mannitol-
fermented foods or be used to synthesize mannitol as food
ingredient or pharmaceutical compound. Heterofermenta-
tive LAB belonging to the genera Leuconostoc, Lactobacil-
lus and Oenococcus produce mannitol from fructose in a
single enzymatic conversion by mannitol 2-dehydrogenase
(MDH), thereby producing less ethanol (no further need for
NAD+ regeneration) and more acetic acid (enabling more
ATP production) (Korakli and Vogel 2003; von Weymarn
2002). In contrast, most homofermentative LAB normally
do not produce mannitol, and its formation is limited to
strains whose ability to regenerate NAD+ is hampered
(Wisselink et al. 2005). A number of heterofermentative
LAB strains have been investigated for their production
of mannitol (Saha and Racine 2011). Saha and Nakamura
(2003) reported on the ability to produce mannitol from fruc-
tose by strains of Lactobacillus brevis, Lactobacillus buch-
neri, Lactobacillus cellobiosus, Lactobacillus fermentum,
Lactobacillus intermedius, Leuconostoc amelibiosum, Leuco-
nostoc citrovorum, Leuconostoc mesenteroides and Leuco-
nostoc paramesenteroides. Recently, Patra et al. (2011)
characterized different mannitol-producing strains of Leuco-
nostoc isolated from a broad range of raw and indigenous
fermented foods. The strain L. intermedius NRRL B-3693
selected by Saha and Nakamura (2003) was able to produce
up to 198 g/l of mannitol from a high fructose concentration
(300 g/l) medium. In a previous work, we evaluated the
production of mannitol by the heterofermentative strains L.
fermentum CRL 573 and Lactobacillus reuteri CRL 1101
using a mixture of glucose and fructose as carbon source
(Rodríguez et al. 2012). However, to produce mannitol cost-
effectively on an industrial scale by fermentation, economical
carbon and nitrogen sources are needed to replace expensive
substrates (Song and Vieille 2009). Argentina is one of the top
ten producer countries of sugarcane (29,950,000 tonnes in
2008) and in particular the province of Tucumán, one of the
main local sugar producers. Here, we employed sugarcane
molasses, a by-product from the local sugar industry, as
Appl Microbiol Biotechnol (2012) 95:991–999
carbon substrate to produce mannitol by the strain L. reuteri
CRL 1101, a LAB species commonly considered as probiotic
(Eaton et al. 2011; Liu et al. 2010; Taranto et al. 2000).
Different culture conditions were investigated as an approach
to improve mannitol production by the studied strain using
this low-cost carbon source.
Materials and methods
Microorganism and culture media
The strain L. reuteri CRL 1101 used in this work was
obtained from the Culture Collection of Centro de Referen-
cia para Lactobacilos (CERELA), San Miguel de Tucumán,
Argentina. Cultures were stored as described by Rodríguez
et al. (2012). L. reuteri CRL 1101 was selected from a
screening for mannitol-producing heterofermentative LAB
strains previously performed (unpublished results).
Cultures were grown in 200 ml of modified MRS con-
taining sugarcane molasses instead of glucose as carbohy-
drate source. As the carbohydrate content and composition
of sugarcane molasses varies greatly depending on the
source, a 1% (w/v) solution of the molasses used in
this study, obtained from a local sugar factory, was
analysed by high-performance liquid chromatography
(HPLC) as described below. Then, a 20% (w/v) stock solution
of sugarcane molasses was prepared as follows: molasses was
weighed, dissolved in distilled water, centrifuged (11,050×g
20 min 4 °C) to remove interfering particles and diluted with
distilled water to the final volume. This solution was added to
modified MRS (twofold concentrated and without glucose) as
well as distilled water to reach a final sugar concentration of
3.0%, 5.0%, 7.5%, or 10.0% (w/v). Finally, the culture media
were sterilized at 121 °C for 20 min. Non-inoculated sterilized
culture medium was incubated at 37 °C for 48 h as a sterility
control.
Fermentation assays
L. reuteri CRL 1101 was transferred twice in MRS containing
3.0% (w/v) of molasses prior to experimental use; overnight
cultures were used as inoculums (2%, v/v). Fermentations
were performed in sealed bottles containing 200 ml of medi-
um and incubated at 37 °C for 48 h. Samples were aseptically
withdrawn at 0, 4, 8, 24 and 48 h and cooled immediately on
ice before further analysis, namely cell growth by determining
the cell counts (colony-forming units per millilitre) through
plating of samples diluted in physiological solution (NaCl
0.85%, w/v) in MRS agar (MRS Britania, Buenos Aires,
Argentina, plus 15 g/l of agar), pH measurements determined
with a digital pH meter (Altronix TPX 1, Brooklyn, NY, USA)
and residual carbohydrates and fermentation end-products
Appl Microbiol Biotechnol (2012) 95:991–999
such as organic acids (both lactic acid and acetic acid), ethanol
and mannitol as described below. Cell growth was not fol-
lowed by optical density measurements due to the interference
of molasses components in the culture medium. When need-
ed, cultures were incubated in MRS supplemented with 7.5%
(w/v) sugarcane molasses at 30 °C or 37 °C and under agita-
tion using a shaker bath at 100 rpm; samples were withdrawn
and analysed as stated before.
Determination of residual sugars, mannitol and organic
acids by HPLC
Mannitol, sugars (glucose, fructose and sucrose), organic acids
(lactic and acetic) and ethanol concentrations were determined
by HPLC. Fermented samples were centrifuged (2,750×g
10 min at 4 °C), and the obtained supernatants were deprotei-
nised in a modified technique according to Vrancken et al.
(2008) as follows: 50 μl of Carrez A reagent [3.6% (w/v) of
K4(Fe(CN)6)·3H2O] and 50 μl of Carrez B reagent [7.2% (w/v)
of ZnSO4·7H2O] were added to 600 μl of cell-free culture
supernatants; then, 100 μl of 0.1 M NaOH was added and
diluted up to 1,000 μl with ultrapure water. Samples were then
centrifuged (14,500×g 5 min at 4 °C), and 50 mg of basic and
acid resins [Amberlite IR 120 (OH) and IR 45 (H), Laboratory
Reagents, Philadelphia, PA, USA] were added, mixed for
10 min and left them at room temperature for 5 min. After
centrifugation (14,500×g 5 min 4 °C), supernatants were
diluted fivefold, filtered (0.2-μm filters, Minisart high-flow;
Sartorius) and kept at −20 °C before analyses. Mannitol and
sugars were analysed using an Aminex HPX-87P column
(Bio-Rad Laboratories Inc., San Francisco, CA, USA) at
85 °C with distilled water as the mobile phase. All compo-
nents were analyzed by HPLC [pump Smartline 100, refrac-
tive index (RI) detector K-2301, Smartline autosampler 3800
Plus, Knauer, Berlin, Germany and Zeltec ZC90 oven,
Buenos Aires, Argentina]. The elution rate was 0.6 ml/min.
Organic acids (lactic and acetic) and ethanol were determined
with an Aminex HPX-87H (Bio-Rad) column at 41 °C using
5 mM H2SO4 as mobile phase and RI detector. All data were
analysed using the Eurochrom Basic Edition for Windows
software.
The efficiency of conversion of fructose into mannitol
was calculated as the amount (millimoles) of mannitol pro-
duced in a defined period (nMtl t0t–nMtl t0 0) divided by the
amount (millimoles) of fructose consumed in the same
period (nFru t0t–nFru t0 0)×100, and was expressed as YMtl (von
Weymarn 2002).
Preparation of cell-free extracts
Cells were harvested from 10 ml of broth at different incu-
bation times (4, 8, 24 and 48 h) by centrifugation (2,750×g
10 min 4 °C) and washed three times with cold 50-mM
993
potassium phosphate buffer (pH 5.5). The wet pellets were
mixed with glass beads (0.1 mm dia, Biospec Products;
Bartlesville, OK, USA) in a 1:2:1 (cells:buffer:beads) ratio.
Then, cells were disrupted using a Mini Bed Beater-8 (Bio-
spec Products) for 8 min (with 2 min interruption on ice
after each minute) at maximum speed. Cell debris, unbroken
cells and glass beads were removed by centrifugation
(12,500×g 8 min 4 °C), and the supernatants were immedi-
ately used for the enzyme assays.
Mannitol 2-dehydrogenase activity
MDH activity was determined according to a modified
method described by Sasaki et al. (2005). Briefly, MDH
was measured spectrophotometrically on a VERSAmax™
Tunable Microplate reader (Sunnyvale, CA, USA); the dis-
appearance of NADPH or NADH (Sigma Chemical Co.,
MO, USA) was monitored by measuring the absorbance at
340 nm (ε340, 6,220 M−1 cm−1) for 5 min. The protein
concentration of cell-free extracts was measured by the
Bradford method (Bradford 1976) using the Bio-Rad Pro-
tein Assay (Bio-Rad Laboratories, USA) following the man-
ufacturer's instructions and using bovine serum albumin as
standard. The cell-free extracts were diluted to obtain a
protein concentration range between 1 and 2 mg prot/ml.
The enzymatic assay was done in a 200-μl volume; the
reaction mixture contained 50 μl 200 mM sodium phos-
phate buffer (pH 5.5), 50 μl 2 mM NADPH or NADH, 50 μl
of ultrapure water and 10 μl of diluted cell-free extract. The
reaction mixture was maintained at 32 °C for 2 min, and the
reaction was started by adding 40 μl of 1 M fructose (Sigma
Chemical Co). One unit (U) of MDH activity was defined as
the amount of enzyme required to catalyze the disappear-
ance (fructose reducing direction) of 1 μmol of NADPH or
NADH per minute under the experimental conditions used.
Specific MDH activity was expressed in units per milligram
cell protein. All enzyme assays were done in triplicate of
two independent experiments.
Statistics analysis
All fermentations were carried out in duplicate, and analyt-
ical determinations were carried out in triplicate; results
were expressed as means with standard deviations.
Results
Growth kinetics and mannitol production using different
molasses concentration
Sugarcane molasses used in this study contained 49% (w/w)
of total sugars, from which sucrose was the main constituent
994
(42%) while glucose and fructose (produced from sucrose
hydrolysis) were present at 3.0% and 4.0%, respectively.
Growth and mannitol production by L. reuteri CRL 1101
were investigated using different molasses concentrations as
carbon source, between 3.0% and 10.0% (w/v) at 37 °C
during 48 h (Fig. 1a–d). The strain could grow well (Δlog
CFU/ml, 2.17–2.48) in all assayed media after an incubation
of 24 h; the highest cell growth (Δlog CFU/ml, 2.48) being
obtained with 7.5% and 10% (w/v) of molasses. Regarding
growth kinetics, maximum cell viability was attained after
8 h in the presence of 3% (w/v) of molasses, while at higher
molasses concentration, maximum growth values were
reached only after 24 h. In all media, culture pH decreased
to values of 4.2–4.4 after 24 h of incubation and remained
stable till the end of the fermentation.
As expected, sugar depletion was faster in the 3% and 5%
(w/v) molasses-supplemented media; in the former one, free-
glucose and -fructose were completely consumed after 8 h
of fermentation, while the available sucrose was exhausted
after 24 h (Fig. 1a). The increase in molasses concentration
(7.5–10%, w/v) in the medium led to a slower sugar deple-
tion, and even after 48 h fermentation, 10% to 13% of the
initial sucrose remained non-fermented. Available glucose
(free and that produced from the hydrolysis of sucrose) was
heterofermentatively converted mainly into lactic and acetic
acids and in a lesser extent into ethanol. This behaviour was
observed at all molasses concentrations used; sugar conver-
sion into organic acids was detected earlier in the presence
of 3% (w/v) of molasses. The highest conversion ratio of
glucose into organic acids was obtained with 7.5% and 10%
(w/v) of molasses. Total fructose (free and that present in
sucrose) was almost completely used as an alternative elec-
tron acceptor and consequently reduced to mannitol.
Mannitol was produced from fructose in all assayed
media from the early growth phase (first 8–10 h of incuba-
tion), and maximum values were reached after 24 h; the
highest mannitol production values were 177.7±26.6 (YMtl,
90%) and 184.5±22.5 mM (YMtl, 89%) in the 7.5% and 10%
(w/v) molasses-supplemented medium, respectively; an in-
crease in the molasses concentration up to 10% (w/v) as well
as in the fermentation period (48 h) did not lead to a
significantly increase in mannitol formation. Similar and
higher amounts of mannitol compared to those of lactic
and acetic acids were obtained when increasing the molasses
concentration to 7.5% and 10% (w/v) (Fig. 1c, d and Table 1).
From the obtained results, a molasses concentration of 7.5%
(w/v) was selected for further studies.
Effect of temperature and agitation on mannitol production
To investigate the effect of temperature on mannitol forma-
tion, L. reuteri CRL 1101 was grown at 30 °C (Fig. 2a, b).
The decrease in temperature from 37 °C to 30 °C resulted in
Appl Microbiol Biotechnol (2012) 95:991–999
Fig. 1 Growth and mannitol production by L. reuteri CRL 1101 in„
modified MRS medium supplemented with different molasses concen-
tration at 37 °C for 48 h; (a) 3%, (b) 5%, (c) 7.5% and (d) 10% (w/v)
molasses
slower cell growth and sugar consumption; after 24 h of
incubation, cell growth was Δlog CFU/ml, 1.9, while the
pH decrease was 1.7 pH units; at this time point, 79% of the
initial sucrose remained non-fermented, while 78% and 48%
of free-glucose and -fructose, respectively, remained avail-
able; no sugar depletion was detected even after 48 h of
fermentation. Consumed glucose was converted into lactic
and acetic acids and ethanol (49.1, 25.6 and 4.13 mM,
respectively), while 82% of the fructose consumed was
reduced into mannitol yielding 48.3±9.7 mM. When the
fermentation was prolonged up to 48 h, higher lactic and
acetic acids and ethanol concentrations (236, 98.9 and
27.1 mM, respectively) were obtained due to higher biomass
formation and concomitant sugar consumption. At this time
(48 h), mannitol production increased up to 217.2±3.9 mM,
a similar value to that obtained when the strain was incu-
bated at 37 °C for 24 h. Fructose was only used as alterna-
tive electron acceptor and reduced into mannitol.
The influence of agitation on the growth and mannitol
production by L. reuteri CRL 1101 was undertaken at 37 °C
(Fig. 3), as a significantly higher mannitol production was
achieved at this temperature than at 30 °C after 24 h fer-
mentation. Different growth kinetics were observed in agi-
tated cultures (Fig. 3a) as compared to static ones (Fig. 1c);
maximum cell growth was observed after 8 h showing a
Δlog CFU/ml, 2.18, slightly lower than the maximum
(Δlog CFU/ml, 2.48) obtained after 24 h in static cultures.
Available sugars were not depleted during fermentation with
the exception of free-fructose, which was completely con-
sumed after 24 h. Organic acids (lactic and acetic) and ethanol
production increased more rapidly under aerated conditions
than in static cultures (Figs. 3b and 1c, respectively).
The increase in mannitol production (211.3±15.5 mM) in
the agitated culture was not statistically significant with
respect to the static one after 24 h; however, the most
interesting feature was that a mannitol value of 144.8 mM,
25 times higher and statistically significant than that
obtained without agitation at the same time point was
reached after only 8 h of incubation. In agitated cultures,
mannitol formation was much higher than lactic and acetic
acids production (Fig. 3b and Table 1).
The stoichiometric parameters for product formation
(Table 1) show that highest sugar (both glucose and
fructose) consumption, mannitol production and ratios
of mannitol to organic acids were obtained with the
best culture conditions (7.5% molasses, 37 °C and agitation)
mentioned before; however, the highest YMtol was obtained in
static cultures.
Appl Microbiol Biotechnol (2012) 95:991–999 995

a) Molasses 3%
300 300

Acetic acid (mM)

300
pH

Ethanol (mM)
Lactic acid (mM)
10 250 250
250

Mannitol (mM)
Fructose (mM)
Glucose (mM)
Sucrose (mM)
200 200
200
8
Log CFU/ml

150 150 150

6 100 100
100

50 50 50
4
0 0 0
0 10 20 30 40 50 0 10 20 30 40 50

Time (h) Time (h)

b) Molasses 5%
300 300 300
pH

Acetic acid (mM)

10

Lactic acid (mM)

250 250 250

Mannitol (mM)

Ethanol (mM)
Fructose (mM)
Glucose (mM)
Sucrose (mM)

200 200 200

8
Log CFU/ml

150 150 150

6 100 100
100

50 50 50
4
0 0 0
0 10 20 30 40 50 0 10 20 30 40 50

Time (h) Time (h)

c) Molasses 7.5%
300 300 300
pH

10

Acetic acid (mM)

Lactic acid (mM)

250 250 250

Mannitol (mM)
Fructose (mM)
Sucrose (mM)

Glucose (mM)

Ethanol (mM)
200 200 200
8
Log CFU/ml

150 150 150

6
100 100 100

50 50 50
4

0 0 0
0 10 20 30 40 50 0 10 20 30 40 50
Time (h) Time (h)

d) Molasses 10%
300 300 300
pH

Acetic acid (mM)

10
Lactic acid (mM)

250 250 250

Mannitol (mM)
Fructose (mM)

Ethanol (mM)
Glucose (mM)
Sucrose (mM)

200 200 200

8
Log CFU/ml

150 150 150

6
100 100 100

50 50 50
4

0 0 0
0 10 20 30 40 50 0 10 20 30 40 50

Time (h) Time (h)

996 Appl Microbiol Biotechnol (2012) 95:991–999

Table 1 Stoichiometric parameters for mannitol and organic acid formation by Lactobacillus reuteri CRL 1101 using sugarcane molasses as
substrate in MRS-supplemented media after 24 h fermentations

Culture conditions Consumption (mMol) Mtl (mMol) YMtl, % Mtl/HLac Mtl/HLac + HAc

Glucose Fructose

Mola 3%, 37 °C 89.1±3.0 89.2±34.3 67.3±312.7 75.5 0.84 0.59

Mol 5%, 37 °C 169.0±322.2 168.0±321.2 108.5±325.4 64.6 0.77 0.56
Mol 7.5%, 37 °C 174.1±335.8 198.0±338.2 177.6±326.6 89.7 1.03 0.75
Mol 10%, 37 °C 174.6±319.2 208.0±323.2 184.5±322.5 88.6 1.08 0.73
Mol 7.5%, 30 °C 67.9±32.8 59.0±312.7 48.0±39.7 82.3 0.98 0.64
Mol 7.5%, 37 °C Ab 201.4±310.0 243.0±35.2 211.3±315.5 86.9 1.24 0.83

Mtl mannitol, Mtl/HLac ratio of mannitol to lactic acid, Mtl/HLac + HAc mannitol ratio to lactic acid + acetic acid
a
Molasses
b
Agitated cultures

Mannitol 2-dehydrogenase activity for the NADPH cofactor rather than NADH for the reduc-
tion of fructose (data not shown). To determine MDH activ-
To investigate if a correlation between mannitol production ity, different reaction temperatures (30 °C, 32 °C, 34 °C and
and MDH activity occurred under the assayed culture con- 37 °C) were used; as the highest MDH activity values were
ditions, MDH measurements were done in cell-free extracts obtained at 32 °C, this temperature was selected for the
obtained from all fermentations at different incubation times enzyme assays (data not shown). On the whole, the highest
(Table 2). NADPH was used instead of NADH, as MDH MDH activity values (3.646–2.064 U/mg cell protein) were
from L. reuteri CRL 1101 showed higher (1.3 times) affinity
a)
300
pH

a) 10
300 250

Fructose (mM)
Sucrose (mM)

Glucose (mM)
pH

10 200
250 8
Fructose (mM)

Log CFU/ml
Glucose (mM)
Sucrose (mM)

200 150
8
Log CFU/ml

6
150 100

6 50
100 4

50 0
4 0 10 20 30 40 50
0 Time (h)
0 10 20 30 40 50
Time (h) b)
300 300
b)
Acetic acid (mM)
Lactic acid (mM)

300 300 250 250

Mannitol (mM)

Ethanol (mM)
Acetic acid (mM)

250 250
Lactic acid (mM)

200 200
Mannitol (mM)

Ethanol (mM)

200 200 150 150

150 150 100 100

100 100
50 50

50 50
0 0
0 10 20 30 40 50
0 0
0 10 20 30 40 50 Time (h)
Time (h)
Fig. 3 Growth and mannitol production by L. reuteri CRL 1101 in
Fig. 2 Growth and mannitol production by L. reuteri CRL 1101 in 7.5% molasses-supplemented MRS medium incubated at 37 °C under
7.5% molasses-supplemented MRS medium at 30 °C for 48 h agitation for 48 h
Appl Microbiol Biotechnol (2012) 95:991–999 997

Table 2 Mannitol 2-
dehydrogenase activity of cell- Culture conditions Specific MDH activity (U/mg cell protein)
free extracts of Lactobacillus
reuteri CRL 1101 grown in Time of incubation (h)
molasses-supplemented MRS
medium under different culture 4 8 24 48
conditions
Mola 3%, 37 °C 3.646±30.052 2.100±30.097 2.340±30.024 2.016±30.067
Mol 5%, 37 °C 2.587±30.098 1.997±30.257 2.122±30.134 1.960±30.086
Mol 7.5%, 37 °C 2.277±30.036 1.847±30.144 1.726±30.308 1.580±30.081
Mol 10%, 37 °C 2.598±30.079 2.094±30.662 2.087±30.083 1.349±30.089
a
Mol 7.5%, 30 °C 0.260±30.086 2.712±30.646 2.685±30.385 1.743±30.075
Molasses
b
Mol 7.5%, 37 °C Ab 2.064±30.145 1.037±30.320 1.075±30.291 0.583±30.166
Agitated culture

found at early growth phases (log phase, 4 h) independently
of the assayed culture conditions; when the fermentation
was carried out at 30 °C, the maximum enzyme activity
value (2.712±0.646 U/mg cell protein) was observed between
8 and 24 h, which corresponded to a slower log phase at this
temperature as compared to those at 37 °C. A decrease in
MDH activity from the log phase was detected in all fermen-
tations. Interestingly, the highest MDH activity value (3.646±
0.052 U/mg cell protein) was obtained in the 3% (w/v)
molasses-MRS supplemented medium, although maximum
mannitol values were obtained at higher molasses concentra-
tions (Fig. 1 and Table 1). Thus, no correlation existed
between the maximum mannitol production values (Table 1)
and the MDH activities (Table 2) for the assayed fermentation
conditions.
Discussion
Mannitol is industrially produced due to its multiple appli-
cations in the food, pharmaceutical, chemical industries and
in medicine; however, a low-efficient chemical process
using glucose/fructose syrup is currently employed which
synthesizes mannitol with the concomitant production of sor-
bitol as by-product.
To produce mannitol in a more efficient way, different
biotechnological approaches using mannitol-producing
LAB have been sought in the last decade. Recently, manni-
tol production by L. reuteri CRL 1101 was studied using
modified MRS containing a glucose/fructose mixture (in a
1:6.5 ratio) as carbon source; the strain produced 91 mM of
mannitol in free-pH fermentations after 24 h (Rodríguez et
al. 2012). Here, mannitol production by the same strain
using sugarcane molasses, a by-product from the sugar
industry, as low-cost energy source was studied. High molas-
ses concentrations ranging from 3% to 10% (w/v), to induce
osmotic stress, were used. L. reuteri CRL 1101 grew well and
was capable of using fructose as an alternative electron accep-
tor and reduced it to mannitol since the log/early stationary
growth phase. This strain efficiently produced mannitol with
values higher than 177 mM and 90% yield when grown in
7.5% (w/v) molasses MRS-supplemented medium at 37 °C
during 24 h. Other few low-cost substrates have been inves-
tigated for mannitol production. Recently, Carvalheiro et al.
(2011) reported on the use of carob syrup (containing sucrose,
fructose, glucose and pinitol in a total sugar concentration of
92 g/l) for mannitol synthesis by different lactobacilli and
Leuconostoc strains. Concentration of mannitol up to 240
and 219 mM were achieved with a Leuconostoc fructosum
strain and L. reuteri DSM 20016, respectively, after an incu-
bation at 37 °C during 24 h. Saha (2006a) evaluated the use of
sugarcane molasses alone and combined with fructose syrup
(3:1) on mannitol production by L. intermedius NRRL-3693
in stirred cultures at controlled pH of 5.0. In the presence of
very high concentration (150 g total sugars per litre) of
molasses alone, this strain produced a maximum amount of
mannitol of 221.7 mM after 40-h incubation, while a value
2.6-fold higher was obtained with a mixture of molasses and
fructose syrup after a 16-h incubation period. For this reason,
Saha (2006a) proposed to use both substrates to attain maxi-
mal mannitol production in a shorter fermentation period.
Increasing the molasses concentration from 7.5% to 10%
(w/v) did not significantly enhance mannitol production by
L. reuteri CRL 1101. Also, Yun and Kim (1998) found that
the strains Lactobacillus sp. Y-107 and Leuconostoc sp. Y-
002 were not able to utilize high concentrations of sugars
above 100 g/l due to the low osmotolerance of these bacte-
ria. In addition, increasing the initial fructose concentration
from 100 to 120 and 140 g/l resulted in decreased mannitol
productivities in Leu. mesenteroides ATCC 9135 due to
both substrate and end-product inhibition of the enzyme
MDH (von Weymarn et al. 2002).
To enhance mannitol production by L. reuteri CRL 1101,
different culture conditions were investigated. Lowering the
incubation temperature from 37 °C to 30 °C provoked a
slower cell growth and concomitant lower organic acid and
mannitol production; only after 48 h of incubation, the
amount of mannitol produced was similar to that found
998
when the strain was incubated at 37 °C for 24 h. von
Weymarn (2002) studied mannitol production by eight het-
erofermentative LAB strains and found that the ability to
produce mannitol from fructose varied markedly among
species. Mannitol yields by L. fermentum NRRL B-1932
were increased from 86 up to 94 mol% when increasing the
temperature from 25 °C to 35 °C using fructose and glucose
(2:1) as carbon sources.
To evaluate whether cultures grown under agitation
favoured mannitol production, agitation of 100 rpm was
applied to cultures containing 7.5% (w/v) molasses incubated
at 37 °C. Mannitol formation was similar in aerated cultures to
static ones after 24-h incubation; however, the most remark-
able result was that high amounts of mannitol (145 mM) were
produced during the early growth phase (8 h of incubation), a
significantly increased value with respect to that obtained in
static cultures at the same time point. In agitated cultures, L.
reuteri CRL 1101 produced comparable amount of mannitol
to that obtained by Saha (2006a) when using molasses as sole
energy source, which was reached using 15% (w/v) molasses
concentration after a long (40 h) fermentation period. Agita-
tion conditions may improve fructose and nutrient availability
for growth and mannitol production by L. reuteri CRL 1101,
especially if cell flocculation occurs as it was previously
observed for this strain (Rodríguez et al. 2012). Also, mannitol
has been reported to accumulate in response to environmental
stresses such as osmotic and oxidative stresses (Wisselink et
al. 2002).
MDH activity during growth of L. reuteri CRL 1101 in
all assayed fermentation conditions was investigated in an
attempt to correlate this enzyme activity with mannitol
production. A few articles on MDH characterization and
purification in heterofermentative LAB have been reported
(Hahn et al. 2003; Korakli and Vogel 2003; Saha 2004;
Sasaki et al. 2005); however, and to our knowledge, MDH
activity was not measured in function of cell growth during
different fermentation conditions. In general, the MDH
activity values obtained here were higher as compared to those
reported by Saha (2004) and Korakli and Vogel (2003), who
found MDH values of 1 U/mg cell protein in crude extracts of
L. intermedius and Lactobacillus sanfranciscensis strains
grown on sucrose and fructose-supplemented MRS medium,
respectively. In our study, the highest MDH activity values
were obtained during the log growth phase in all fermentations
assayed, which agrees with mannitol formation during the log
and early stationary phases and the cell growth-associated
mannitol production reported for this strain in free-pH fer-
mentations (Rodríguez et al. 2012). In contrast, maximum
mannitol production values by L. reuteri CRL 1101 were not
correlated with the highest MDH activity (3.646±0.052 U/mg
cell protein) detected in 3% (w/v) molasses-supplemented
medium at 37 °C after 24 h. Other studies, such as mdh gene
expression in different culture conditions are needed to better
Appl Microbiol Biotechnol (2012) 95:991–999
understand and explain this fact. The NADPH-dependency of
MDH of L. reuteri CRL 1101 is in accordance with the high
NADPH dependency for the MDH of L. reuteri ATCC 53608
and L. intermedius NRRL B-3693 (Saha 2004; Sasaki et al.
2005).
The use of sugarcane molasses as low-cost carbon source
represents an important approach to further design an inex-
pensive, more competitive culture medium for the biotech-
nological production of mannitol. The evaluation of other
medium components (i.e. nitrogen, vitamin and salts) to
replace the MRS medium and to ensure good cell growth
and mannitol formation by L. reuteri CRL 1101 are still
needed. Controlled-pH fermentations using molasses-
supplemented medium are currently under study, as this
strain produces higher mannitol amounts under controlled-
pH (5.0) than in free-pH cultures when using glucose and
fructose as carbon sources (Rodríguez et al. 2012)
L. reuteri CRL 1101, a promising candidate to produce
mannitol as food ingredient, efficiently produces mannitol
from sugarcane molasses. To our knowledge, this is the first
detailed report on mannitol synthesis by a L. reuteri strain
using sugarcane molasses as energy source.
Acknowledgements We acknowledge the financial support of
CONICET (PIP2010-0062), FONCyT (Préstamo BID PICT2008-
933) and CIUNT from Argentina. M. E. Ortiz is recipient of a doctoral
fellowship from CONICET, Argentina.
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